PROJECT SUMMARY Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the third leading cause of cancer death in both men and women in the United States. Our understandings of plausible risk factors of CRC are based on paradigms drawn from epidemiological studies. Meat consumption and cigarette smoking are risk factors for CRC. Carcinogenic N-nitroso compounds (NOCs), heterocyclic aromatic amines (HAAs), and polycyclic aromatic hydrocarbons (PAHs) occur in processed meats or cooked red meat, and some of these chemicals arise in tobacco smoke. Heme also can catalyze the endogenous formation of nitroso compounds and lipid peroxides. Red meat consumption and cigarette smoking are risk factors of CRC; however, unambiguous physico-chemical evidence of chemicals in meat and tobacco that form DNA adducts in the colorectum are lacking, and the underlying mechanisms of CRC initiation are not known. The paucity of accessible frozen tissue has precluded the use of DNA adducts as biomarkers in epidemiology studies and limited our understanding about the roles of chemical exposures in DNA damage and CRC etiology. In contrast, formalin fixed paraffin embedded (FFPE) tissue is frequently available. Until now, the screening of DNA adducts in FFPE tissue has been restricted to immunohistochemical (IHC) analysis. However, the specificity of many antibodies, even monoclonal antibodies, for DNA adducts is uncertain as they can cross- react with other DNA lesions or cellular components, leading to errors in identification and quantification. Moreover, the screening of DNA adducts, by IHC, requires the costly development of many antibodies. We recently established a novel technology to retrieve DNA from rodent or human FFPE tissues employing mild conditions to reverse the crosslinks of DNA formed with formalin, while preserving the structures of the DNA adducts of aristolochic acids, NOCs, aromatic amines, HAAs, and PAHs. We propose the use of FFPE colorectal tissues as biospecimens to assess the impact of chemical exposures in the etiology of CRC, by measuring DNA adducts with specific mass spectrometry (MS)-based methods. There is astonishingly little biomarker data on the chemicals that form DNA adducts in the colorectum. The goal of our proposal is to refine our newly developed technology to retrieve DNA adducts from FFPE colorectal tissues of rodents following exposures to genotoxicants in tobacco and meat, and then employ the technology to identify DNA adducts in FFPE specimens of CRC patients who are smokers and/or meat-eaters. Adducts will be measured by nanoflow chromatography with high resolution accurate mass spectrometry. The successful validation of our method will greatly advance our understanding about the toxicants in tobacco and diet that da- mage colorectal DNA. Moreover, the DNA adduct biomarkers can be linked to mutations induced in cancer driver genes, and to genetic variants in carcinogen metabolism and DNA repair enzymes that impact the risk of CRC.